1. Field of the Invention
The present invention relates generally to the fields of neurobiology and developmental biology. More specifically, the present invention relates to the identification of novel neuregulin splice variant isoforms.
2. Description of the Related Art
Traumatic injury of adult mammalian peripheral nerve results in degeneration of axon segments and myelin distal to the injury site with concomitant Schwann cell dedifferentiation and proliferation. These changes in Schwann cell morphology are essential for subsequent axonal regeneration (Hall and Gregson, 1977; Pellegrino et al., 1986; Fawcett and Keynes, 1990; Nadim et al., 1990) and are accompanied by increased Schwann cell expression of molecules promoting neurite sprouting [e.g., neurotrophic factors and cell adhesion molecules; reviewed in Fawcett and Keynes, 1990; Fu and Gordon, 1997]. The signals responsible for repressing myelin protein synthesis, inducing expression of molecules supportive of axonal regeneration, and stimulating Schwann cell mitogenesis in injured nerve are poorly understood. It is likely, however, that these signaling molecules include several members of the neuregulin (NRG) family of growth and differentiation factors.
The neuregulin (NRG) family of growth and differentiation factors is thought to form a complex network of intercellular signaling molecules mediating multiple important developmental, maintenance and regenerative functions throughout the nervous system. For instance, neuregulins are highly expressed by sensory and motor neurons during development (Chen et al., 1994; Falls et al., 1993; Ho et al., 1995; Marchionni et al., 1993) and have been implicated as axon-derived signals influencing the differentiation, survival and proliferation of associated Schwann cells during this same period (reviewed in (Topilko et al., 1996; Lemke, 1996)). Neuregulins are also highly potent mitogens for neonatal Schwann cells in vitro (Brockes et al., 1980; Goodearl et al., 1993; Levi et al., 1995) and repress expression of myelin protein zero (P0) and myelin basic protein in these same cells (Cheng and Mudge, 1996). Furthermore, axon-associated NRGs are a component of the xe2x80x9caxon-associated mitogenxe2x80x9d found on the neurites of neonatal dorsal root ganglion (DRG) neurons (Morrissey et al., 1995). Based on these developmental and in vitro observations, it is hypothesized that neuregulins, potentially released from the injured axon, similarly induce the Schwann cell dedifferentiation and proliferation during the Wallerian degeneration which follows traumatic injury of peripheral nerve and which is essential for subsequent axonal regeneration (Hall and Gregson, 1977; Pellegrino et al., 1986; Fawcett and Keynes, 1990; Nadim et al., 1990).
These molecules are indeed expressed with the temporal and spatial distribution expected for postaxotomy mediators of Schwann cell proliferation and/or other effects in axotomized rat sciatic nerve (Carroll et al., 1997). However, Schwann cells themselves apparently produce neuregulin, a finding consistent with recent reports of neuregulin expression by cultured neonatal Schwann cells in vitro (Raabe et al., 1996; Rosenbaum et al., 1997). Furthermore, the dorsal root ganglia (DRG) sensory and spinal cord motor neurons projecting into the sciatic nerve express the erbB receptors necessary for neuregulin responsiveness during embryogenesis and adulthood. Also, recombinant neuregulin is a survival factor for embryonic day 15 spinal cord motor neurons in vitro. It is therefore likely that neuregulin signaling proceeds bidirectionally between these cell types or that Schwann cell- and neuron-derived neuregulins act in an autocrine fashion.
Since astrocytes, oligodendrocytes and many populations of central nervous system (CNS) neurons similarly express both neuregulins and neuregulin receptors, these same possibilities may need to be considered in the brain. Given the potential complexity of neuregulin signaling among glia and neurons, the question arises as to how neuregulin signaling might be compartmentalized or otherwise regulated. This control may be facilitated, in part, by the synthesis of distinct forms of neuregulin by each expressing cell type. Cloning of neuregulin family members (Wen et al., 1992; Marchionni et al., 1993; Carroll et al., 1997; Falls et al., 1993; Ho et al., 1995; Carroll et al., 1997; Yang et al., 1998) demonstrated these molecules to be structurally diverse proteins translated from alternatively spliced mRNAs transcribed from a single locus. Neuregulins may be divided into three subfamilies, each defined by their unique N terminus and known as the heregulin (HRG)/neu differentiation factor (NDF)/mesenchymal, glial growth factor (GGF) and sensory and motor neuron-derived factor [SMDF; also known as cysteine-rich domain (CRD)-neuregulin] subfamilies.
The structures of various members of the neu differentiation factor subfamily have been thoroughly studied. The seven known neu differentiation factor isoforms are synthesized as either directly secretable forms or as transmembrane precursors requiring proteolytic cleavage for release (Wen et al., 1994). These proteins possess distinct epidermal growth factor (EGF)-like domains (xcex1 and xcex2 isoforms) resulting in differences in receptor affinity (Wen et al., 1994) and ability to induce biological effects (Marikovsky et al., 1995; Pinkas-Kramarski et al., 1996).
The EGF-like domain, which consists of a common region fused to either xcex1- or xcex2-domains, is essential for biologic activity. Truncated xcex2-neuregulin molecules containing only the EGF-like domain bind to the neuregulin receptor with an affinity similar to that of the full-length factor (Holmes et al., 1992; Peles et al., 1993) and are capable of inducing a variety of biologic responses (Holmes et al., 1992; Peles et al., 1993; Chu et al., 1995; Levi et al., 1995; Syroid et al., 1996).
In spite of their similar structures, neuregulin xcex1 and xcex2 EGF-like domains are not functionally equivalent; xcex2-neuregulins have an affinity for erbB receptors an order of magnitude greater than xcex1-neuregulins (Wen et al., 1994). Furthermore, xcex1-neuregulins are nonmitogenic for some, but not all, cell types which proliferate in response to xcex2-neuregulins (Pinkas-Kramarski et al., 1996).
Further variability in other regions may alter glycosylation (Wen et al., 1994), protease-mediated release from the cell membrane (Wen et al., 1994) and direct signaling by transmembrane precursors (Wang et al., 1998). In addition to the unique amino termini (the functions of which are currently unknown), the mesenchymal and GGF (but not the SMDF) neuregulin subfamilies contain an immunoglobulin-like domain (Ben-Baruch and Yarden, 1994; Peles and Yarden, 1993; Ho et al., 1995) mediating neuregulin interactions with cell surface glycoproteins, with resultant concentration and specific localization of the factor (Sudhalter et al., 1996). Splice variants in the glial growth factor and mesenchymal neuregulin subfamilies also may contain serine and threonine-rich spacer domains which serve as the site of o- and n-linked glycosylation (Wen et al., 1994; Carroll et al., 1997); this glycosylation is non-essential for biologic activity and the precise function(s) of this region is as yet unknown.
Neuregulins may be synthesized as either transmembrane precursors or directly secretable forms. This distinction depends upon the juxtamembrane domain, which is immediately C terminal to the EGF-like domain. Four juxtamembrane domains, designated 1 to 4, have been identified in the rat. In this regard, the xe2x80x983xe2x80x99 juxtamembrane domain is notable in that it, unlike other juxtamembrane domains, contains a termination codon, thus leading to truncation of the factor and synthesis in a directly secretable form. In all other neuregulin isoforms, the juxtamembrane domain is followed by a transmembrane domain which anchors the factor in the cell membrane and is itself coupled to one of three possible cytoplasmic domains (designated a, b, and c) (Wen et al., 1994). The cytoplasmic domains are highly conserved between species, suggesting an essential function (Wen et al., 1994); indeed, it has been recently reported that neuregulin cytoplasmic domains bind LIM kinase 1, suggesting that neuregulin transmembrane precursors are capable of transmitting signals into the interior of the cell synthesizing these proteins (Wang et al., 1998).
Whether members of the glial growth factor and sensory and motor neuron-derived factor subfamilies demonstrate the same degree of structural diversity described for the NDF subfamily has not yet been determined. It is highly likely that the neuregulin isoforms present in injured peripheral nerve represent a diverse population of previously unknown glial growth factor and sensory and motor neuron-derived factor splice variants. The neuregulins selectively induced in axotomized peripheral nerve coincident with the onset of Schwann cell DNA synthesis belong predominantly to the glial growth factor subfamily, while neuregulins of both the glial growth factor and sensory and motor neuron-derived factor subfamilies are expressed in DRG and spinal cord (Carroll et al., 1997).
The prior art is deficient in the lack of knowledge about the sensory and motor neuron-derived factor (SMDF) and glial growth factor (GGF) neuregulin splice variants expressed in the nervous system. The present invention fulfills this longstanding need and desire in the art.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.
Reverse transcription-polymerase chain reaction (RT-PCR) analyses suggest that axotomized sciatic nerve, DRG and spinal cord all contain complex mixtures of neuregulin isoforms, potentially representing a large number of previously undescribed splice variants with novel functional characteristics. Consequently, an exhaustive cloning approach was used to identify the neuregulin isoforms expressed in surgically transected rat sciatic nerve, postaxotomy lumbar dorsal root ganglia, postaxotomy lumbar spinal cord and JS1 schwannoma cells, a rat line mimicking at least some characteristics of primary cultures of neonatal rat Schwann cells. The structures of cDNAs encoding six SDMF splice variants and four GGF isoforms are described here, representing both directly secreted proteins and transmembrane precursors. These proteins demonstrate extensive structural variability in multiple regions, suggesting they are functionally distinct.
Whether neuregulin isoforms are predominantly expressed in axotomized peripheral nerve and postaxotomy DRG and spinal cord; (2) whether distinct neuronal subpopulations in the latter two tissues express specific neuregulin splice variants; and, (3) what was the distribution of each group of transcripts was examined. In addition, the biochemical properties of particular neuregulin isoforms, was characterized. These results suggest that neuregulins acting in injured peripheral nerve are part of a complex and tightly regulated network of autocrine/paracrine signals. The operation of this network may rely, in part, on the synthesis of structurally and functionally distinct neuregulin splice variants by specific cellular populations within these tissues.
In one embodiment of the current invention, a cDNA encoding SMDFxcex21a, a novel sensory and motor neuron-derived factor (SMDF) splice variant isoform cDNA, is provided. The instant invention is also directed to an isolated. SMDFxcex21a protein and a plasmid allowing expression of SMDFxcex21a in a cell.
In another embodiment of the current invention, a cDNA encoding a second novel sensory and motor neuron-derived factor (SMDF) splice variant isoform, SMDFxcex12a, is described. The instant invention is directed to a plasmid containing this cDNA sequence and the regulatory elements necessary for expression of SMDFxcex12a in a cell and is also directed to an isolated SMDFxcex12a protein.
A further embodiment of the instant invention is a partial amino acid sequence of SMDF splice variant protein, SMDFxcex12b. The current invention includes an SMDFxcex12b protein containing this sequence as well as and a cDNA and plasmid encoding it.
Yet another embodiment of the instant invention comprises partial amino acid and nucleotide sequences of SMDF splice variant proteins SMDFxcex22, SMDFxcex23, and SMDFxcex24. The instant invention is directed to isolated proteins containing these amino acid sequences as well as cDNA molecules and plasmids encoding them.
Yet another embodiment of the instant invention comprises partial amino acid and nucleotide sequences of glial growth factor splice variant proteins GGFxcex21a, GGFxcex22, GGFxcex23, and GGFxcex24. The instant invention is directed to isolated proteins containing these amino acid sequences as well as cDNA molecules and plasmids encoding them.
A further embodiment of the instant invention is a method of treating condition of nerve dysfunction comprising the step of administering an effective dose of SMDFxcex21a, SGGFxcex12a, SMDFxcex12b, SMDFxcex22, SMDFxcex23, SMDFxcex24, GGFxcex21a, GGFxcex22, GGFxcex23, or GGFxcex24. Such a method of treatment is likely to be useful in the treatment of demyelinating diseases such as multiple sclerosis, nerve injuries such as spinal cord and peripheral nerve injuries and neuropathies, neurodegenerative diseases such as Alzheimer""s disease and Parkinson""s disease, and motor neuron diseases such as ALS and Werdnig-Hoffman disease.